Tumor suppressor gene polypeptides and related nucleic acids, host cells, compositions, and methods of use in inhibition of cell growth, modulation of gene expression, and enhancement of immune-response inducing effect of a vaccine

ABSTRACT

A polypeptide consisting essentially of the amino acid sequence NCEHFVTYCRYG (SEQ ID NO: 1), ENCEHFVNELRYG (SEQ ID NO: 2), NCEHFVNELRYG (SEQ ID NO: 3), RNCEHFVAQLRYG (SEQ ID NO: 4), NCEHFVAQLRYG (SEQ ID NO: 5), or NCEHFVTYLRYG (SEQ ID: 6), or a variant of any of the foregoing, a composition comprising same, a nucleic acid consisting essentially of a nucleotide sequence encoding the amino acid sequence of any of SEQ ID NOS: 1-6 or a variant thereof, a composition comprising same, a host cell comprising an above-described nucleic acid molecule, a method of inhibiting cancerous cell growth comprising contacting a collection of cancerous cells with a cell growth-inhibiting effective amount of one or more of the foregoing, a method of modulating gene expression comprising contacting a collection of cells with a gene expression-modulating effective amount of at least one above-described nucleic acid or a composition comprising same, and a method of enhancing the immune response-inducing effect of a vaccine, which method comprises adding to the vaccine at least one above-described polypeptide, or a variant thereof.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a purified or isolated polypeptideconsisting essentially of the amino acid sequence NCEHFVTYCRYG (SEQ IDNO: 1), ENCEHFVNELRYG (SEQ ID NO: 2), NCEHFVNELRYG (SEQ ID NO: 3),RNCEHFVAQLRYG (SEQ ID NO: 4), NCEHFVAQLRYG (SEQ ID NO: 5), orNCEHFVTYLRYG (SEQ ID NO: 6), a purified or isolated nucleic acidconsisting essentially of a nucleotide sequence encoding anaforementioned polypeptide, related compositions and host cells, amethod of inhibiting cell growth, a method of modulating geneexpression, and a method of enhancing the immune response-inducingeffect of a vaccine.

BACKGROUND OF THE INVENTION

The American Cancer Society estimates the lifetime risk that anindividual will develop cancer is 1 in 2 for men and 1 in 3 for women.The development of cancer, while still not completely understood, can beenhanced as a result of a variety of risk factors. For example, exposureto environmental factors (e.g., tobacco smoke) might triggermodifications in certain genes, thereby initiating cancer development.Alternatively, these genetic modifications may not require an exposureto environmental factors to become abnormal. Indeed, certain mutations(e.g., deletions, substitutions, etc.) can be inherited from generationto generation, thereby imparting an individual with a geneticpredisposition to develop cancer.

The desire of cancer research is the identification of a therapyeffective on several different types of cancers. Yet, despite extensiveresearch into the disease, effective cancer therapeutics remain elusivefor the medical community. Clinicians have realized limited success withthe current standard therapies, chemotherapy, radiation therapy, andsurgery, inasmuch as each therapy has inherent limitations. Chemotherapyand radiation therapy cause extensive damage to normal, healthy tissue,despite efforts to target such therapy to abnormal tissue (e.g.,tumors). Surgery can be effective in removing masses of cancerous cells;however, even the most talented surgeon cannot ensure complete removalof affected tissue, nor are all tumors in an anatomical locationamenable to surgical removal. The limitations of existing therapies arereflected in the 60% 5-year relative survival rate for all cancerscombined (Cancer Facts & Figures 2001, The American Cancer Society, NewYork, N.Y.).

Systemic toxicity of drugs is one of the most serious problems of cancerchemotherapy and frequently is dose-limiting. The appearance of variousclasses of multiple drug resistant cancerous cells renders even gooddrugs ineffective, since they are expelled from the tumor cells (Ling,Cancer Chemother. Pharmacol. 40: Suppl, S3-S8 (1997)). Variousstrategies have been used to get around one or both of thesedifficulties, but they still are among the most intractable problems ofcancer therapy. Targeting of drugs specifically to tumor cells has beenthe goal of many studies. Various protein toxins conjugated tomonoclonal antibodies directed to specific tumor antigens have shownsome promise as drugs (Pastan, Biochim. Biophys. Acta 1333: C1-C6(1997)), but severe problems, such as the development of neutralizingantibodies (Chen et al., Gene Ther. 2: 116-123 (1995)), have limited theeffectiveness of the method.

Another promising approach is to use cellular receptors for growthfactors (Kihara et al., Cancer Res. 55: 71-77 (1985); Carpenter, Curr.Opin. Cell Biol. 5: 261-264 (1993); and Lemaristre et al., Breast CancerRes. Treat. 32: 97-103 (1994)), cytokines (Strom et al., Annu. Rev. Med.44: 343-353 (1993); and Waldmann et al., Ann. Intern. Med. 116: 148-160(1992)), or hormones (Roth et al., Anticancer Drug Des. 10: 655-666(1994); and Rink et al., Proc. Natl. Acad. Sci. 93: 15063-15068 (1996))as targets to deliver cytotoxic moieties to the receptor-bearing cells.In this approach, the receptor binds to a ligand that is conjugated to atoxic moiety, resulting in receptor-mediated endocytosis, wherein theligand-toxic moiety conjugate is internalized, along with the receptor,by the targeted cell. Once inside the cell, the conjugate is susceptibleto lysosomal proteases that cleave the linkage between the ligand andtoxin, resulting in the release of the toxin from the conjugate. Throughthis approach, the delivery of a drug to specific cell populations canbe achieved.

Other efforts to decrease treatment-related toxicities and enhancespecific cytotoxicity of cancer treatment have focused on immunotherapyto generate a specific anticancer immunity, metabolic pathways to targetspecifically cancerous cells, and identification of genes responsiblefor the malignant transformation of normal cells into cancerous cells.Clinicians also have looked to the delivery of therapeutic nucleic acidsequences as a possible alternative to existing cancer therapies. Thelocal production of therapeutic agents at biologically-significantlevels in target sites in vivo, thereby reducing the toxicity to normaltissues, addresses some of the limitations associated with conventionaltherapy.

Accordingly, there remains a need for a composition suitable for use intreating a variety of cancer types in a patient, as well as a method fordelivering the composition to treat cancer. The present inventionprovides such a composition and method. This and other objects andadvantages of the invention, as well as additional inventive features,will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a purified or isolated polypeptideconsisting essentially of the amino acid sequence NCEHFVTYCRYG (SEQ IDNO: 1), ENCEHFVNELRYG (SEQ ID NO: 2), NCEHFVNELRYG (SEQ ID NO: 3),RNCEHFVAQLRYG (SEQ ID NO: 4), NCEHFVAQLRYG (SEQ ID NO: 5), orNCEHFVTYLRYG (SEQ ID NO: 6), or a variant of any of the foregoing, anyone of which is optionally part of a fusion protein or conjugated to anagent that increases the potency and/or specificity of the polypeptide.Also provided is a composition comprising one or more of theaforementioned polypeptides and a carrier, excipient or adjuvant.

Also provided by the present invention is a purified or isolated nucleicacid consisting essentially of a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, or a variant of any of the foregoing,optionally as part of an encoded fusion protein, and optionally in theform of a vector, which is optionally targeted to a membrane receptor.The nucleic acid can have a peptidic backbone when not in the form of avector. A composition comprising one or more of the aforementionednucleic acids and a carrier, excipient or adjuvant is also provided.

A host cell comprising an above-described purified or isolated nucleicacid molecule is also provided. The nucleic acid can have a peptidicbackbone when not in the form of a vector.

Further provided is a method of inhibiting cancerous cell growth. Themethod comprises contacting a collection of cancerous cells with a cellgrowth-inhibiting effective amount of one or more of the following:

-   -   (i) at least one above-described purified or isolated        polypeptide, which can be the same or different,    -   (ii) a composition comprising (i) and a carrier, excipient or        adjuvant,    -   (iii) at least one above-described purified or isolated nucleic        acid, which can be the same or different, and    -   (iv) a composition comprising (iii) and a carrier, excipient or        adjuvant, whereupon the growth of the collection of cancerous        cells is inhibited. The method optionally further comprises        separately contacting the collection of cancerous cells with an        anti-cancer agent in the same manner or a different manner,        surgical removal of the collection of cancerous cells when in        vivo, and/or radiation.

Still further provided is a method of modulating gene expression. Themethod comprises contacting a collection of cells with a geneexpression-modulating effective amount of one or more of the following:

-   -   (i) at least one above-described purified or isolated nucleic        acid, which can be the same or different and which has a        peptidic backbone, and    -   (ii) a composition comprising (i) and a carrier, excipient or        adjuvant, whereupon gene expression in the collection of cells        is modulated.

Also still further provided is a method of enhancing the immuneresponse-inducing effect of a vaccine. The method comprises adding tothe vaccine at least one above-described purified or isolatedpolypeptide, which can be the same or different, whereupon the immuneresponse-inducing effect of the vaccine is enhanced.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the surprising and unexpecteddiscovery that tumor suppressor gene polypeptides (TSGP) candramatically inhibit the proliferation of cancerous cells. The TSGP haveanti-proliferative, anti-tumor and cytostatic properties. In thisregard, the present invention provides a purified or isolatedpolypeptide consisting essentially of the amino acid sequenceNCEHFVTYCRYG (SEQ ID NO: 1), ENCEHFVNELRYG (SEQ ID NO: 2), NCEHFVNELRYG(SEQ ID NO: 3), RNCEHFVAQLRYG (SEQ ID NO: 4), NCEHFVAQLRYG (SEQ ID NO:5), or NCEHFVTYLRYG (SEQ ID NO: 6). The term “isolated” as used hereinmeans having been removed from its natural environment. The term“purified” as used herein means having been increased in purity, wherein“purity” is a relative term, and not to be construed as absolute purity.In each embodiment provided herein, a letter indicates the standardamino acid designated by that letter. Additionally, in accordance withconvention, all amino acid sequences provided herein are given from leftto right, such that the first amino acid is amino-terminal and the lastamino acid is carboxyl-terminal. The polypeptide preferably comprises anamino end and a carboxyl end. The polypeptide can comprise D-aminoacids, L-amino acids or a mixture of D- and L-amino acids. The D-form ofthe amino acids, however, is particularly preferred, since a polypeptidecomprised of D-amino acids is expected to have a greater retention ofits biological activity in vivo, given that the D-amino acids are notrecognized by naturally occurring proteases.

The polypeptides (and variants thereof as described herein below) can beprepared by any of a number of conventional techniques. The polypeptidecan be isolated or purified from a naturally occurring source or from arecombinant source. For instance, in the case of recombinantpolypeptides, a DNA fragment encoding a desired peptide can be subclonedinto an appropriate vector using well-known molecular genetic techniques(see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory, 1989) and other references citedherein under “EXAMPLE”). The fragment can be transcribed and thepolypeptide subsequently translated in vitro. Commercially availablekits also can be employed (e.g., such as manufactured by Clontech, PaloAlto, Calif.; Amersham Life Sciences, Inc., Arlington Heights, Ill.;InVitrogen, San Diego, Calif., and the like). The polymerase chainreaction optionally can be employed in the manipulation of nucleicacids.

Such polypeptides also can be synthesized using an automated peptidesynthesizer in accordance with methods known in the art. Alternately,the polypeptide (including the variant peptides) can be synthesizedusing standard peptide synthesizing techniques well-known to those ofskill in the art (e.g., as summarized in Bodanszky, Principles ofPeptide Synthesis, (Springer-Verlag, Heidelberg: 1984)). In particular,the polypeptide can be synthesized using the procedure of solid-phasesynthesis (see, e.g., Merrifield, J. Am. Chem. Soc., 85, 2149-54 (1963);Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987); andU.S. Pat. No. 5,424,398). If desired, this can be done using anautomated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC)or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups andseparation of the polypeptide from the resin can be accomplished by, forexample, acid treatment at reduced temperature. Thepolypeptide-containing mixture then can be extracted, for instance, withdimethyl ether, to remove non-peptidic organic compounds, and thesynthesized polypeptide can be extracted from the resin powder (e.g.,with about 25% w/v acetic acid). Following the synthesis of thepolypeptide, further purification (e.g., using high performance liquidchromatography (HPLC)) optionally can be done in order to eliminate anyincomplete polypeptides or free amino acids. Amino acid and/or HPLCanalysis can be performed on the synthesized polypeptide to validate itsidentity. For other applications according to the invention, it may bepreferable to produce the polypeptide as part of a larger fusionprotein, either by chemical conjugation, or through genetic means, suchas are known to those skilled in the art.

The polypeptides of SEQ ID NOS: 1-6 are preferred. However, conservativeand neutral amino acid substitutions can be introduced into thepolypeptides. In this regard, the present invention also provides apurified or isolated variant of an above-described polypeptide. Whentumor suppressor activity is desired, NCEHFV and RYG, i.e., amino acids1-6 and 10-12 of SEQ ID NOS: 1, 3, 5 and 6, and amino acids 2-7 and11-13 of SEQ ID NOS: 2 and 4, should remain unchanged. Desirably, theleucine at amino acid position 9 in SEQ ID NOS: 3, 5 and 6 also shouldremain unchanged for tumor suppressor activity. The variant comprisesone, two or three conservative or neutral amino acid substitutions,provided that amino acids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and9 in SEQ ID NO: 2, amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1and 8 in SEQ ID NO: 4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 inSEQ ID NO: 6 remain unchanged. Where amino acid 7 can be altered,preferably amino acid 7 is uncharged when tumor suppressor activity isdesired. Where amino acid 8 is altered, it can have other than apositive charge, such as a neutral or negative charge or hydrophobicity,and still maintain tumor suppressor activity. Additionally oralternatively, the variant comprises one, two or three amino acidadditions at the N-terminus and/or C-terminus. Preferably, not more thana total of 1, 2 or 3 amino acids are added. Desirably, the variant hasactivity characteristic of the unaltered polypeptide, optionally to agreater or lesser extent, but not negated.

Alterations of the native amino acid sequence to produce variantpolypeptides can be done by a variety of means known to those skilled inthe art. For instance, amino acid substitutions can be convenientlyintroduced into the polypeptides at the time of synthesis.Alternatively, site-specific mutations can be introduced by ligatinginto an expression vector a synthesized oligonucleotide comprising themodified site. Alternately, oligonucleotide-directed, site-specificmutagenesis procedures can be used, such as disclosed in Walder et al.,Gene, 42, 133 (1986); Bauer et al., Gene, 37, 73 (1985); Craik,Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and4,737,462.

It is within the skill of the ordinary artisan to select synthetic andnaturally-occurring amino acids that effect conservative or neutralsubstitutions for any particular naturally-occurring amino acids. Theskilled artisan desirably will consider the context in which anyparticular amino acid substitution is made, in addition to consideringthe hydrophobicity or polarity of the side-chain, the general size ofthe side chain and the pK value of side-chains with acidic or basiccharacter under physiological conditions. For example, lysine, arginine,and histidine are often suitably substituted for each other, and moreoften arginine and histidine. As is known in the art, this is becauseall three amino acids have basic side chains, whereas the pK value forthe side-chains of lysine and arginine are much closer to each other(about 10 and 12) than to histidine (about 6). Similarly, glycine,alanine, valine, leucine, and isoleucine are often suitably substitutedfor each other, with the proviso that glycine is frequently not suitablysubstituted for the other members of the group. This is because each ofthese amino acids are relatively hydrophobic when incorporated into apolypeptide, but glycine's lack of an α-carbon allows the phi and psiangles of rotation (around the α-carbon) so much conformational freedomthat glycinyl residues can trigger changes in conformation or secondarystructure that do not often occur when the other amino acids aresubstituted for each other. Other groups of amino acids frequentlysuitably substituted for each other include, but are not limited to, thegroup consisting of glutamic and aspartic acids; the group consisting ofphenylalanine, tyrosine and tryptophan; and the group consisting ofserine, threonine and, optionally, tyrosine. Additionally, theordinarily skilled artisan can readily group synthetic amino acids withnaturally-occurring amino acids.

If desired, the polypeptides of the invention (including variantpolypeptides) can be modified, for instance, by glycosylation,amidation, carboxylation, or phosphorylation, or by the creation of acidaddition salts, amides, esters, in particular C-terminal esters, andN-acyl derivatives of the polypeptides of the invention. Thepolypeptides also can be modified to create polypeptide derivatives byforming covalent or noncovalent complexes with other moieties inaccordance with methods known in the art. Covalently-bound complexes canbe prepared by linking the chemical moieties to functional groups on theside chains of amino acids comprising the polypeptides, or at the N- orC-terminus. Desirably, such modifications and conjugations do notadversely affect the activity of the polypeptides (and variantsthereof). While such modifications and conjugations can have greater orlesser activity, the activity desirably is not negated and ischaracteristic of the unaltered polypeptide.

Thus, in this regard, the present invention also provides a fusionprotein comprising an above-described isolated or purified polypeptide(or variant thereof) and a conjugate comprising an above-describedisolated or purified polypeptide (or variant thereof) and an agent thatincreases the potency and/or specificity of the polypeptide or variantthereof. Examples of such an agent include, but are not limited to, aligand or an antibody (or fragment thereof) for a cell-surface receptoror molecule, the expression of which is specific to a particular type ofcell, in particular a cancerous cell, such as a cell of melanoma, animmunoprotein sequence known to be selectively expressed (e.g.,melanoma-associated antigens, such as MART-1/Melan-A (Schutz et al.,Cancer Gene Ther. 8: 655-661 (2001)), or overexpressed on melanoma cells(see, e.g., Brown et al., Curr. Oncol. Rep. 3: 344-352 (2001)), animport peptide, a protein transduction domain, an antisense molecule,such as an antisense molecule that is specific for a gene encoding aretinoic acid receptor (RAR) or an over-expressed receptor, a ribozymeto a gene that is expressed only in a particular type of cell oroverexpressed in a particular type of cell, e.g., a cancerous cell, suchas an antisense oncogene, e.g., c-fos, a homeobox gene, and an RAR gene,e.g., RAR-β, which has aberrant expression in melanoma, an anti-canceragent, and the like.

Ligands include, for example, a protein or polypeptide ligand, when thetarget is a cell-surface receptor, a steroid, when the target is asteroid receptor, and the like. Analogs of targeting moieties thatretain the ability to bind to a defined target also can be used. Inaddition, synthetic targeting moieties can be designed, so as to fit aparticular epitope on a cell surface, for example. Alternatively, thepolypeptide can be encapsulated in a liposome comprising on its surfacea targeting moiety, such as a ligand or an antibody or immunologicallyreactive fragment thereof.

Examples of cancer-specific, cell-surface receptors include erbB-2,erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL4 (lymphoma andleukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin(gliomas), tumor vasculature integrins, and the like. Preferredcancer-specific, cell-surface receptors include erbB-2 and tumorvasculature integrins, such as CD11a, CD11b, CD11c, CD18, CD29, CD51,CD61, CD66d, CD66e, CD106, and CDw145.

The antibody can be a polyclonal antibody or a monoclonal antibody or animmunologically reactive fragment of either of the foregoing.Alternatively, an engineered immunoprotein can be used. Antibodies,immunologically reactive fragments thereof, and immunoprotiens can begenerated in accordance with methods known in the art, including thoseset forth in the references listed herein under “EXAMPLE.”

There are a number of antibodies to cancer-specific, cell-surfacemolecules and receptors that are known. C46 Ab (Amersham) and 85A12 Ab(Unipath) to carcino-embryonic antigen, H17E2 Ab (ICRF) to placentalalkaline phosphatase, NR-LU-10 Ab (NeoRx Corp.) to pan carcinoma, HMFC1Ab (ICRF) to polymorphic epithelial mucin, W14 Ab to B-human chorionicgonadotropin, RFB4 Ab (Royal Free Hospital) to B-lymphocyte surfaceantigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) tohuman melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP7059588), and the like. ScAbs can be developed, based on suchantibodies, using techniques known in the art (see for example, Bind etal., Science 242: 423-426 (1988), and Whitlow et al., Methods 2(2):97-105 (1991)).

Import peptides and protein transduction domains can be used to improvetransport of the polypeptide across the cell membrane, particularly whenthe cell membrane is selective or demonstrates poor permeability. See,e.g., Park et al. (J. Gen Virol. 83: 1173-1181 (2002)) with respect toprotein transduction domains, and Lanford et al. (Cell 15: 5875-5882(1986)) and Adam et al. (Cell 66: 837-847 (1988)) with respect tonuclear localization sequences. See, also, Morris et al., Nature Biotech19: 1173-1175 (2001), and Gilon et al., Biopolymers 31: 745-750 (1991).

The antisense molecule preferably is at least about 20 nucleotides inlength, and the ribozyme preferably comprises at least about 20continuous nucleotides complementary to a target sequence on each sideof the active site of the ribozyme. The nucleic acid sequence introducedin antisense suppression generally is substantially identical to atleast a portion, preferably at least about 20 contiguous nucleotides, ofthe gene to be targeted, but need not be identical. A vector expressingan antisense molecule can, thus, be designed such that the inhibitoryeffect applies to other proteins within a family of genes exhibitinghomology or substantial homology to the target gene. The introducedsequence also need not be full-length relative to either of the primarytranscription product or fully processed mRNA. Generally, higherhomology can be used to compensate for the use of a shorter sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and homology of non-coding segments will be equallyeffective. As an alternative to antisense suppression, interfering RNAcan be used to achieve the same effect by a different mechanism ofaction.

Ribozymes can be designed such that they specifically pair withvirtually any target RNA and cleave the phosphodiester backbone at aspecific location, thereby functionally inactivating the target RNA. Incarrying out this cleavage, the ribozyme is not itself altered and is,thus, capable of recycling and cleaving other molecules, making it atrue enzyme. The inclusion of ribozyme sequences with antisense RNAsconfers RNA-cleaving activity upon them, thereby increasing the activityof the constructs. The design and use of target RNA-specific ribozymesis described in Haseloff et al., Nature 334: 585-591 (1988). Preferably,the ribozyme comprises at least about 20 contiguous nucleotidescomplementary to the target sequence on each side of the active site ofthe ribozyme.

Alternatively, the polypeptide or variant thereof can be conjugated to areporter group, including, but not limited to a radiolabel, afluorescent label, such as fluorescein, an enzyme (e.g., that catalyzesa colorimetric or fluorometric reaction), a substrate, a solid matrix,or a carrier (e.g., biotin or avidin). Methods of conjugation are knownin the art. In addition, conjugate kits are commercially available. Whenthe polypeptide or variant thereof is labeled with flourescein, suchlabeling can be done by Sigma Genosys according to their labelingprotocol for 5(6)-carboxyfluorescein. In order to preserve thepolypeptide's tumor suppressor activity, maintain the ability of theconjugated fluorophore to emit at its optimal wavelength, and maintainthe ability of the polypeptide to cross cellular membranes, thefluorophore should be placed at the N-terminus of the polypeptide, whichhas the requisite free amine, rather than within or at the C-terminus.Detectably labeled polypeptides can be used to determine whether or notcancerous cells can be treated with the polypeptides of the presentinvention having tumor suppressor activity. Detection of the labeledpolypeptide within the nucleus of a cell indicates that the cell issusceptible to treatment with a present inventive polypeptide.Detectably labeled polypeptides also can be used to identify othermolecular targets in the development of cancer, such as melanoma.

In view of the foregoing, the present invention also provides acomposition comprising (i) an above-described purified or isolatedpolypeptide (or variant thereof) and (ii) a carrier, excipient oradjuvant. The polypeptide or variant thereof can be optionally part of afusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide or variant as set forth above. Thepolypeptides can serve as neoadjuvants to decrease margins of primarytumors in surgical settings.

When the polypeptide consists essentially of the amino acid sequence ofSEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or a variant of any of theforegoing, the composition can further comprise a retinoid. Retinoidsinclude agents that bind to the retinoic acid receptor, such as9-cis-retinoic acid, 4-hydroxy-retinoic acid, all trans-retinoic acid,(E)-4-[2-(5,6,7,8-tetrahydro-2-naphthylenyl)-1-propenyl]-benzoic acid,and3-methyl-(E)₄-[2-(5,6,7,8-tetrahydro-2-naphthylenyl)-1-propenyl]-benzoicacid).

Optionally, the composition further comprises an anti-cancer agent.Examples of anti-cancer agents include those set forth herein below.

Preferably, the carrier is pharmaceutically acceptable. The carrier canbe any of those conventionally used and is limited only bychemico-physical considerations, such as solubility and lack ofreactivity with the active agent of the present invention, and by theroute of administration. It is preferred that the pharmaceuticallyacceptable carrier be one which is chemically inert to the active agentand one which has no detrimental side effects or toxicity under theconditions of use. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those ordinarily skilled in the art and are readilyavailable to the public. Typically, the composition, such as apharmaceutical composition, can comprise a physiological salinesolution; dextrose or other saccharide solution; or ethylene, propylene,polyethylene, or other glycol. The TSGP are soluble in water.

Also in view of the foregoing, the present invention provides a purifiedor isolated nucleic acid consisting essentially of a nucleotide sequenceencoding an above-described amino acid sequence (or variant thereof).The terms “purified” and “isolated” have the meaning set forth above.The term “nucleic acid” as used herein means a polymer of DNA or RNA,(i.e., a polynucleotide), which can be single-stranded ordouble-stranded, synthesized or obtained from natural sources, and whichcan contain natural, non-natural or altered nucleotides. With respect tothe isolated or purified nucleic acid of the present invention, it ispreferred that no insertions, deletions, inversions, and/orsubstitutions are present in the nucleic acid. When tumor suppressoractivity is desired, the nucleic acid should encode amino acids 1-6 and10-12 of SEQ ID NOS: 1, 3, 5 and 6, and amino acids 2-7 and 11-13 of SEQID NOS: 2 and 4. Desirably, the nucleic acid also should encode aminoacid 9 in SEQ ID NOS: 3, 5 and 6 when tumor suppressor activity isdesired. However, it may be suitable in some instances for the isolatedor purified nucleic acid to encode one or more conservative and/orneutral amino acid substitutions, provided that amino acids 8 and 9 inSEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2, amino acids 7 and8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO: 4, and amino acid 7in SEQ ID NO: 5 remain unchanged. Where amino acid 7 can be altered inone of the present inventive polypeptides and tumor suppressor activityis desired, preferably the nucleic acid encodes an amino acid atposition 7 that is uncharged. Where amino acid 8 can be altered in oneof the present inventive polypeptides and tumor suppressor activity isdesired, the nucleic acid can encode an amino acid at position 8 thathas other than a positive charge, such as a neutral or negative charge,or hydrophobicity. In addition to conservative and neutral amino acidsubstitutions, nucleotides can be added at the 5′ and/or 3′ end of thenucleic acid such that amino acids are added to the N and/or C terminusof the encoded polypeptide. Preferably, not more than up to a total of1, 2 or 3 amino acids are added. Desirably, the encoded variant hasactivity characteristic of the unaltered polypeptide, optionally to agreater or lesser extent, but not negated. A variety of techniques usedto synthesize the oligonucleotides of the present invention are known inthe art. See, for example, Lemaitre et al., Proceedings of the NationalAcademy of the Sciences 84: 648-652 (1987).

The nucleic acid can have a peptidic backbone, preferably when it is notpart of a vector. Such nucleic acids are referred to as peptidic nucleicacids or PNAs, functional properties of which optimize modulation ofgene expression include length, target sequence and G/C content (see,e.g., Doyle et al., Biochem 40: 53-64 (2001)). PNAs can be obtained fromApplied Systems (Framingham, Mass.), or synthesized using the t-BOCstrategy of solid phase synthesis (see, e.g., Langel et al., Int. J.Pept. Pro Res. 39: 516-522 (1992)) or another synthesis method (see,e.g., Christensen et al., J. Pept. Sci. 3: 175 (1995); Haaima et al.,Angew. Chem., Int. Ed. Engl. 35: 1939 (1996); and Paschl et al.,Tetrahedron Lett. 39: 4707 (1998)). Parity of the PNA can bedemonstrated by HPLC. The molecular mass can be determined by massspectrometry. PNAs for the polypeptides of SEQ ID NOS: 1-6 and variantsthereof will range from 9 to about 36 bases. PNA also can be labeledwith a detectably labeled probe, such as those known in the art, e.g.,biotin, FITC and anthracene, and used as a probe to identify or assessthe mechanism or molecular interaction of TSGPs with DNA.

In view of the above, the present invention also provides a vectorcomprising an above-described isolated or purified nucleic acidmolecule, optionally as part of an encoded fusion protein. The vectorcan be targeted to a membrane receptor if so desired. A nucleic acidmolecule as described above can be cloned into any suitable vector andcan be used to transform or transfect any suitable host. The selectionof vectors and methods to construct them are commonly known to personsof ordinary skill in the art and are described in general technicalreferences (see, in general, “Recombinant DNA Part D,” Methods inEnzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987) andthe references cited herein under “EXAMPLE”). Desirably, the vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant or animal) into which the vector isto be introduced, as appropriate and taking into consideration whetherthe vector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid as described above or a portion thereofligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColE 1, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

One of ordinary skill in the art will appreciate that any of a number ofvectors known in the art are suitable for use in the invention. Suitablevectors include those designed for propagation and expansion or forexpression or both. Examples of suitable vectors include, for instance,plasmids, plasmid-liposome complexes, and viral vectors, e.g.,parvoviral-based vectors (i.e., adeno-associated virus (AAV)-basedvectors), retroviral vectors, herpes simplex virus (HSV)-based vectors,and adenovirus-based vectors. Any of these expression constructs can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994).Examples of cloning vectors include the pUC series, the pBluescriptseries (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEXseries (Clonetech, Palo Alto, Calif.). Bacteriophage vectors, such asλGT10, λGT11, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can beused. Examples of plant expression vectors include pBI101, pBI101.2,pBI110.3, pBI121 and pBIN19 (Clonetech, Palo Alto, Calif.). Examples ofanimal expression vectors include pEUK-C1, pMAM and pMAMneo (Clonetech).

An expression vector can comprise a native or normative promoteroperably linked to an isolated or purified nucleic acid as describedabove. The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the skill in theart. Similarly, the combining of a nucleic acid molecule as describedabove with a promoter is also within the skill in the art.

Optionally, the isolated or purified nucleic acid molecule, upon linkagewith another nucleic acid molecule, can encode a fusion protein. Thegeneration of fusion proteins is within the ordinary skill in the art(see, e.g., references cited under “Example”) and can involve the use ofrestriction enzyme or recombinational cloning techniques (see, e.g.,Gateway™ (Invitrogen, Carlsbad, Calif.)). See, also, U.S. Pat. No.5,314,995.

A targeting moiety also can be used in the contact of a cell with anabove-described isolated or purified nucleic acid or variant thereof. Inthis regard, any molecule that can be linked with the nucleic aciddirectly or indirectly, such as through a suitable delivery vehicle,such that the targeting moiety binds to a membrane receptor, such as acell-surface receptor or nuclear membrane receptor, or other molecule,can be used. The targeting moiety can bind to a cell through a receptor,a substrate, an antigenic determinant or another binding site on thesurface of the cell. Examples of a targeting moiety include an antibody(i.e., a polyclonal or a monoclonal antibody), an immunologicallyreactive fragment of an antibody, an engineered immunoprotein and thelike, a protein (target is receptor, as substrate, or regulatory site onDNA or RNA), a polypeptide (target is receptor), a peptide (target isreceptor), a steroid (target is steroid receptor), and the like. Analogsof targeting moieties that retain the ability to bind to a definedtarget also can be used. In addition, synthetic targeting moieties canbe designed, such as to fit a particular epitope. Alternatively, thenucleic acid can be encapsulated in a liposome comprising on its surfacethe targeting moiety. Examples of cell-surface receptors, ligands, andantibodies are set forth above.

The targeting moiety includes any linking group that can be used to joina targeting moiety to, in the context of the present invention, anabove-described nucleic acid molecule. It will be evident to one skilledin the art that a variety of linking groups, including bifunctionalreagents, can be used. The targeting moiety can be linked to the nucleicacid by covalent or non-covalent bonding. If bonding is non-covalent,the conjugation can be through hydrogen bonding, ionic bonding,hydrophobic or van der Waals interactions, or any other appropriate typeof binding.

Thus, the present invention further provides a composition comprising(i) one or more of an above-described purified or isolated nucleic acidor variant thereof, optionally as part of an encoded fusion protein, and(ii) a carrier, excipient or adjuvant. The nucleic acid is optionally inthe form of a vector, which is optionally targeted to a membranereceptor. When the nucleic acid is not in the form of a vector, thenucleic acid optionally can have a peptidic backbone. When the nucleicacid encodes the amino acid of SEQ ID NO: 4, the amino acid of SEQ IDNO: 5, the amino acid of SEQ ID NO: 6, or a variant of any one or moreof the foregoing, the composition can further comprise a retinoid. Thecomposition can further comprise an anti-cancer agent, examples of whichare set forth herein below. Suitable carriers, excipients and adjuvantsare known in the art and described herein. Suitable compositions are asset forth herein.

A host cell comprising an above-described purified or isolated nucleicacid or variant thereof, optionally as part of an encoded fusionprotein, and optionally in the form of a vector, which is optionallytargeted to a membrane receptor, is also provided. When the nucleic acidor variant thereof is not in the form of a vector, the nucleic acidoptionally can have a peptidic backbone.

Examples of host cells include, but are not limited to, a human cell, ahuman cell line, E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, andN. crassa, E. coli, in particular E. coli TB-1, TG-2, DH5α, XL-Blue MRF′(Stratagene), SA2821 and Y1090 are preferred hosts.

A method of inhibiting cancerous cell growth is also provided. Themethod comprises contacting a collection of cancerous cells with a cellgrowth-inhibiting effective amount of one or more of the following:

-   -   (i) at least one purified or isolated polypeptide or variant        thereof as described herein, which can be the same or different,        and which can be optionally targeted to an agent that increases        the potency and/or specificity of the polypeptide or variant        thereof,    -   (ii) a composition comprising (i) and a carrier, excipient or        adjuvant, and, optionally, an anti-cancer agent, and, when the        polypeptide consists essentially of the amino acid sequence of        SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, optionally, a        retinoid,    -   (iii) at least one purified or isolated nucleic acid or variant        thereof as described herein, which can be the same or different,        optionally in the form of a vector, which is optionally targeted        to a cell-surface receptor on the collection of cells, with the        proviso that, when the purified or isolated nucleic acid is not        in the form of a vector, the purified or isolated nucleic acid        can have a peptidic backbone, and    -   (iv) a composition comprising (iii) and a carrier, excipient or        adjuvant, and, optionally, an anti-cancer agent, and when the        nucleic acid encodes the amino acid of SEQ ID NO: 4, SEQ ID NO:        5 or SEQ ID NO: 6, optionally retinoic acid. “Membrane receptor”        is used generically to refer to a cell-surface receptor,        cell-surface molecule, nuclear membrane receptor, and nuclear        membrane molecule. The method can further comprise separately        contacting the collection of cells with an anti-cancer agent,        which can be the same or different, in the same manner or a        different manner, surgical removal of the collection of cancer        cells when in vivo, and/or radiation, and the like.

A “cell growth-inhibiting effective amount” is an amount sufficient toinhibit the growth, i.e., increase in cellular mass or cellularreplication or proliferation, of the collection of cells, such ascancerous cells, to any degree or to inhibit the onset of cancer.Preferably, the collection of cells is in vivo. A particularly preferredcollection of cells in vivo is melanoma.

Preferably, the collection of cells is in a mammal. For purposes of thepresent invention, mammals include, but are not limited to, the orderRodentia, such as mice, and the order Logomorpha, such as rabbits, theorder Carnivora, including Felines (cats) and Canines (dogs), the orderArtiodactyla, including Bovines (cows) and Suines (pigs), the orderPerssodactyla, including Equines (horses), the order Primate, Ceboid, orSimoid (monkeys), or the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

Any suitable method can be used to contact the collection of cells andwill depend on whether the cells are in vitro or in vivo. If thecollection of cells is in vivo, the method used to contact thecollection of cells will also depend on whether the collection of cellsis on the surface of the body or internal to the body. Suitable routesof in vivo administration are known in the art and include, oral,topical, epidermal, intradermal, transdermal, systemic, intravenous,perenteral, intraperitoneal, and the like. One skilled in the art willappreciate that suitable methods of administering the active agents ofthe present invention or composition thereof to an animal, e.g., amammal such as a human, are known, and, although more than one route canbe used to administer a particular composition, a particular route canprovide a more immediate and more effective reaction than another route.

Desirably, the active agent is administered in the form of apharmaceutically acceptable composition (see, e.g., Remington'sPharmaceutical Sciences, 17th ed., (Mack Publishing Company,Philadelphia, Pa.: 1985), and Langer, Science, 249, 1527-1533 (1990))suitable for topical administration. Such compositions are known in theart. A formulation suitable for topical application can be in the formof creams, ointments, or lotions in which the inhibitor can be mixedwith conventional oleoginous or emulsifying excipients.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the conjugate dissolved indiluents, such as water or saline, (b) capsules, sachets or tablets,each containing a predetermined amount of the active ingredient, assolids or granules, (c) suspensions in an appropriate liquid, and (d)suitable emulsions.

Tablet forms can include one or more of lactose, mannitol, cornstarch,potato starch, microcrystalline cellulose, acacia, gelatin, colloidalsilicon dioxide, croscarmellose sodium, talc, magnesium stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, such carriersas are known in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect the desiredresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular polypeptide, nucleic acid,or composition and the condition of the animal (e.g., human), as well asthe body weight of the animal (e.g., human) to be treated. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side effects that might accompany the administration of aparticular polypeptide, nucleic acid, or composition. A suitable dosagefor internal administration is 0.01 to 100 mg/kg per day. A preferreddosage is 0.01 to 35 mg/kg per day. A more preferred dosage is 0.05 to 5mg/kg per day. A suitable concentration of the conjugate inpharmaceutical compositions for topical administration is 0.05 to 15%(by weight). A preferred concentration is from 0.02 to 5%. A morepreferred concentration is from 0.1 to 3%. The TSGP is effective at 2 μMin vitro over a 48-hour period and at 5 μg/kg per injection in vivo. Asfew as three injections can result in inhibition of tumor growth. Thesize of a tumor can be maintained with daily injections. Ultimately, theattending physician will decide the dosage and the amount of conjugateof the present invention with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, polypeptide, nucleic acid, orcomposition to be administered, route of administration, whetherindividual cells, a tissue, an organ or an organism is being contacted,the particular site being treated, and degree of inhibition needed.

A retinoid is preferably administered topically or systemically,depending on the type of cancer. For example, if the cancer is melanoma,desirably, the retinoid is administered topically. If the cancer isinternal to the body, the retinoid is desirably administeredsystemically.

The method can be used in combination with other known treatmentmethods, such as radiation, surgery, or the administration of otheractive agents, such as adjuvants or other anti-cancer agents and theirprodrugs. Examples of cytotoxic agents and their prodrugs includegenistein, okadaic acid, 1-β-D-arabinofuranosyl-cytosine,arabinofuranosyl-5-aza-cytosine, cisplatin, carboplatin, actinomycin D,asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil,cyclohexyl-chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin,etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogenmustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine,5-fluorouracil, 5-fluorocytosine, adriamycin, cyclophosphamide,methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogenagents such as tamoxifen, intracellular antibodies against oncogenes,the flavonol quercetin, Guan-mu-tong extract, retinoids such asfenretinide, nontoxid retinoid analogues such asN-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as limonene,perillyl alcohol and sobrerol. The anti-cancer agent can be achemotherapeutic agent, e.g., a polyamine or an analogue thereof.Examples of therapeutic polyamines include those set forth in U.S. Pat.Nos. 5,880,161, 5,541,230 and 5,962,533; Saab et al., J. Med. Chem. 36:2998-3004 (1993); Bergeron et al., J. Med. Chem. 37(21): 3464-3476(1994); Casero et al., Cancer Chemother. Pharmacol 36: 69-74 (1995);Bernacki et al., Clin. Cancer Res. 1: 847-857 (1995); Bergeron et al.,J. Med. Chem. 40: 1475-1494 (1997); Gabrielson et al., Clinical CancerRes. 5: 1638-1641 (1999); and Bergeron et al., J. Med. Chem. 43: 224-235(2000), which can be administered alone or in combination with otheractive agents, such as anti-cancer agents, e.g.,cis-diaminedichloroplatinum (II) and1,3-bis(2-chloroethyl)-1-nitrosourea.

Other actions that can be taken include the administration oftumor-infiltrating lymphocytes that express cytokines, RGD-containingpeptides and proteins, which can be administered following surgery, orlipophilic drug-containing liposomes to which are covalently conjugatedmonoclonal antibodies for targeting to cancer cells, adherence to a lowfat diet, moderate physical exercise and hormonal modulation. Forprostate cancer, anti-testosterone agents can be used as well as aninhibitor of cellular proliferation produced by prostatic stromal cellsand C-CAM, an epithelial cell adhesion molecule.

When the collection of cancerous cells is in the form of a tumor,preferred routes of administration include intratumoral and peritumoralroutes of administration. A preferred manner of administering a separateanti-cancer agent is by targeting to a cancer cell. In this regard,examples of cancer-specific, cell-surface molecules are as set forthabove.

Generally, when an above-described polypeptide is administered to ananimal, such as a mammal, in particular a human, it is preferable thatthe polypeptide is administered in a dose of from about 1 to about 1,000micrograms of the polypeptide per kg of the body weight of the host perday when given parenterally. However, this dosage range is merelyexemplary, and higher or lower doses may be chosen in appropriatecircumstances. For instance, the actual dose and schedule can varydepending on whether the composition is administered in combination withother pharmaceutical compositions, or depending on interindividualdifferences in pharmacokinetics, drug disposition, and metabolism. Oneskilled in the art easily can make any necessary adjustments inaccordance with the necessities of the particular situation.

If desired, the half-life of the polypeptide can be increased byconjugation to soluble macromolecules, such as polysaccharides, orsynthetic polymers, such as polyethylene glycol, as described, forinstance, in U.S. Pat. Nos. 5,116,964, 5,336,603, and 5,428,130.Alternately, the polypeptides can be “protected” in vesicles composed ofsubstances such as proteins, lipids (for example, liposomes),carbohydrates, or synthetic polymers. If liposomes are employed,liposome delivery can be carried out as described in U.S. Pat. No.5,468,481, or using liposomes having increased transfer capacity and/orreduced toxicity in vivo (see, e.g., international patent application WO95/21259 and the references cited therein). Furthermore, polypeptidescan be administered in conjunction with adenovirus (preferablyreplication-deficient adenovirus) to allow the intracellular uptake ofthe polypeptides by adenoviral-mediated uptake of bystander molecules(e.g., as described in international patent application WO 95/21259).Similarly, a fusion of an above-described polypeptide and an antibody(or an antigenically reactive fragment thereof) that recognizes a cellsurface antigen; etc. as described can be employed to deliver theresultant fusion protein to a specific target cell or tissue (e.g., asdescribed in U.S. Pat. No. 5,314,995).

Those of ordinary skill in the art can easily make a determination ofthe amount of an above-described isolated and purified nucleic acidmolecule to be administered to an animal, such as a mammal, inparticular a human. The dosage will depend upon the particular method ofadministration, including any vector or promoter utilized. For purposesof considering the dose in terms of particle units (pu), also referredto as viral particles, it can be assumed that there are 100particles/pfu (e.g., 1×10¹² pfu is equivalent to 1×10¹⁴ pu). An amountof recombinant virus, recombinant DNA vector or RNA genome sufficient toachieve a tissue concentration of about 10² to about 10¹² particles perml is preferred, especially of about 10⁶ to about 10¹⁰ particles per ml.In certain applications, multiple daily doses are preferred. Moreover,the number of doses will vary depending on the means of delivery and theparticular recombinant virus, recombinant DNA vector or RNA genomeadministered.

A method of modulating gene expression is also provided by the presentinvention. By “modulating” is meant an increase or decrease in geneexpression as desired. The method comprises contacting a collection ofcells with a gene expression-modulating effective amount of one or moreof the above-described nucleic acids (or variants thereof), which can bethe same or different, optionally in the form of a composition furthercomprising a carrier, excipient or adjuvant, provided that the purifiedor isolated nucleic acid has a peptidic backbone. By “geneexpression-modulating effective amount” is mean an amount of the nucleicacid (or variant thereof) or composition comprising same that caneffectively increase or decrease the expression of a target gene asdesired. When the composition comprises a nucleic acid encoding SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or a variant of any of theforegoing, the composition preferably further comprises a retinoid asdescribed herein. Preferably, the collection of cells is in vivo, suchas in a mammal. Preferably, the cells are cancerous cells, in particularcells of melanoma, in which case the gene is RAR β or high mobilitygroup (HMG) I (Y), both of which have a retinoic acid response elementin their promoter regions, and the expression of the gene isupregulated. Methods of contacting a collection of cells and suitablecompositions for use in the method are as described herein. While notwishing to be bound to any particular theory, it is believed that thepresent inventive polypeptides can modulate gene expression by bindingto a retinoic acid response element, e.g., DR5, in the promoter regionof a gene.

Also provided is a method of enhancing the immune response-inducingeffect of a vaccine. The method comprises adding to the vaccine at leastone above-described purified or isolated polypeptide (or variantthereof), which can be the same or different, optionally as part of afusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide or variant thereof.

EXAMPLES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1997),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1998),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y. (1988),-   Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999),-   Hoffman, Cancer and the Search for Selective Biochemical Inhibitors,    CRC Press (1999),-   Pratt, The Anticancer Drugs, 2nd edition, Oxford University Press,    NY (1994), and-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd    edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y. (1989).

The following example serves to illustrate the present invention andshould not be construed as in any way limiting its scope.

TSGPs were designed based on the published 12-amino acid sequencehomology of human lecithin retinol acyltransferase (LRAT), human Rev(H-Rev), and human tazarotene-induced gene 3 (H-TIG3). Lyophilized TSGPswere synthesized by SIGMA-GENOSYS (The Woodlands, Tex.) and solubilizedin sterile ultra pure water (KD Medical, Columbia, Md.) to achieve a 10mM stock. Aliquots were prepared for one-time use and stored at −80° C.

Melanoma cells were purchased from the American Type Culture Collection(ATCC, Rockville, Md.) and maintained as specified by the ATCC.Specifically, the melanoma cells were Hs939.T, designated as a malignantmelanoma cell line and the matched tumor pairs [688(A).T, 688(B).T] and[WM-115, WM-266-4]. Each pair was obtained from a single patient andconsists of the primary site tumor cells and cells of a tumor thatmetastasized from the primary site. Normal human primary epidermalmelanocytes (HFSC/2) were obtained from the Yale Skin Diseases ResearchCenter (New Haven, Conn.) and, upon arrival, were maintained inmelanocyte growth medium from Clonetics (San Diego, Calif.). The purityof the normal human primary epidermal melanocyte cultures was achievedby growth media restriction of contaminating fibroblasts andkeratinocytes. All cell types were identified as adult tissue in origin.

Cultures were seeded, in duplicate, in 6-well dishes to achieve 60-70%confluence overnight. The next day, 10 mM stocks of TSGPs were dilutedin sterile ultra pure water for a working solution of 10 μM. Monolayerswere either mock-treated, untreated, or treated with TSGP to a finalconcentration of 1 μM. Fresh dilutions of TSGPs were prepared daily andadded at time zero and 24 hrs. Untreated cells were harvested at 24 and48 hrs and treated cells were harvested at 48 hrs by trypsinization andresuspensed in growth media and a 1:1 solution of 0.1% trypan blue. Themonolayer from each well and its respective medium were counted for liveand dead cells using a hemacytometer. Expected cell proliferation wasassessed by comparing the cell number at 24 and 48 hrs in the untreatedgroup.

Daily treatment over 48 hrs of cultures of human melanoma cells, Hs939.T, with 1 μM TSGP:H-TIG3 decreased the number of proliferating cellsby 65.6%, while treatment with 1 μM TSGP:H-Rev107 reduced the number ofproliferating cells by 44%. Importantly, daily treatment of cultures ofproliferating normal human melanocytes with 1 μM TSGPs had nosignificant effect on cell proliferation.

When cultures of Hs 939.T were treated daily over a 48 hour-period with1 μM TSGP:HTIG3 and 1 μM 9-cis retinoic acid (RA), the observed decreasein cell number with TSGP:H-TIG3 alone was not enhanced. Combinationtreatment with TSGP:H-Rev107 was enhanced so that the reduction in cellnumber was increased from 44% to 57%. Either TSGP alone or incombination with 9-cis RA was more effective than 9-cis RA alone.

When 1 μM TSGPs were incubated with cultures of primary site tumorcells, Hs 688(A).T and WM-115, cell proliferation was decreased 45% and57%, respectively, within 48 hours. However, cultures of metastasizedtumors from the primary tumors 688(B).T and WM-266-4 resulted in a 10%and 25%, respectively, reduction in cell number.

Parallel cultures of treated and untreated cells were harvested bylysing the monolayers with SDS lysis buffer and probed with antibodiesto HMG I (Y) and RAR β, (Santa Cruz Biotechnology, Santa Cruz, Calif.).Protein (20 μg) was loaded on a 4-12% Bis-Tris SDS-PAGE (Novex,Carlsbad, Calif.; Invitrogen, Carlsbad, Calif.) reducing gel, separatedby electrophoresis, and transferred onto a 0.2 μm polyvinyldienedifluoride membrane (PVDF). Blots were probed with antibody to HMG I (Y)and RAR β. Signal detection was performed using polyclonal goatanti-mouse conjugated to horseradish peroxidase (Biorad, Hercules,Calif.) in combination with the West Pico SuperSignal chemiluminescentkit (Pierce, Rockford, Ill.). Actin was detected with a polyclonalrabbit anti-actin (Chemicon International, Temecula, Calif.). Specificsignal was quantified by ImageQuant Analysis (Molecular Dynamics,Sunnyvale, Calif.). Treatment groups were normalized againstdensitometry units for untreated, normal human proliferatingmelanocytes, set at a value of 1.

Untreated melanoma cells, Hs 939.T, were downregulated for RAR β and HMGI (Y) relative to proliferating normal human melanocytes (HFSC/2). Whenmelanoma cultures of Hs 939.T were treated with the TSGP:H-TIG3, theprotein expression levels of HMG I (Y) and RAR β were returned tonormal, while treatment with TSGP:H-Rev107 resulted in a near normalreturn of each transcription factor level.

WM-115 cells from benign cutaneous melanoma (ATCC catalog no. 1675) weregrown in normal growth medium and then harvested, washed, and suspendedin phosphate-buffered saline. Five six-week old Balb/c nu/nu female micewere injected subcutaneously with 5-6×10⁵ cells/animal. Mice werechecked weekly for weight and tumor growth. In order to determineantitumor activity, tumors were allowed to grow at least 20 mm³, atwhich point intratumoral injections of peptide (SEQ ID NO: 3 or SEQ IDNO: 4) at 5 μg/kg animal were given three times over a two-week period.Measurements were taken every other day after peptide treatment untilthe animal was terminated. To assess whether the peptides were able tomaintain the tumor size, tumors were treated as described above, thentumors were allowed to re-establish growth. Intratumoral injections weregiven and tumor measurements taken daily, over a 5 day period. Overall,a 3 to 13 fold reduction in tumor size was observed upon treatment. Thepolypeptides not only inhibited tumor growth after three injections, butmaintained the size of the tumor with daily injections.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A purified or isolated polypeptide consisting essentially of theamino acid sequence NCEHFVTYCRYG (SEQ ID NO: 1), optionally as part of afusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide.
 2. A purified or isolatedpolypeptide consisting essentially of the amino acid sequenceENCEHFVNELRYG (SEQ ID NO: 2), optionally as part of a fusion protein orconjugated to an agent that increases the potency and/or specificity ofthe polypeptide.
 3. A purified or isolated polypeptide consistingessentially of the amino acid sequence NCEHFVNELRYG (SEQ ID NO: 3),optionally as part of a fusion protein or conjugated to an agent thatincreases the potency and/or specificity of the polypeptide.
 4. Apurified or isolated polypeptide consisting essentially of the aminoacid sequence RNCEHFVAQLRYG (SEQ ID NO: 4), optionally as part of afusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide.
 5. A purified or isolatedpolypeptide consisting essentially of the amino acid sequenceNCEHFVAQLRYG (SEQ ID NO: 5), optionally as part of a fusion protein orconjugated to an agent that increases the potency and/or specificity ofthe polypeptide.
 6. A purified or isolated polypeptide consistingessentially of the amino acid sequence NCEHFVTYLRYG (SEQ ID NO: 6),optionally as part of a fusion protein or conjugated to an agent thatincreases the potency and/or specificity of the polypeptide.
 7. Apurified or isolated variant of: (i) a polypeptide consistingessentially of the amino acid sequence of NCEHFVTYCRYG (SEQ ID NO: 1),(ii) a polypeptide consisting essentially of the amino acid sequence ofENCEHFVNELRYG (SEQ ID NO: 2), (iii) a polypeptide consisting essentiallyof the amino acid sequence of RNCEHFVAQLRYG (SEQ ID NO: 4), (iv) apolypeptide consisting essentially of the amino acid sequence ofNCEHFVAQLRYG (SEQ ID NO: 5), or (v) a polypeptide consisting essentiallyof the amino acid sequence of NCEHFVTYLRYG (SEQ ID NO: 6), whichcomprises one, two or three conservative or neutral amino acidsubstitutions, provided that amino acids 1-6 and 8-12 in SEQ ID NO: 1,amino acid 1-9 and 11-13 in SEQ ID NO: 2, amino acids 1-8 and 11-13 inSEQ ID NO: 4, amino acids 1-7 and 9-12 in SEQ ID NO: 5, and amino acids1-6 and 8-12 in SEQ ID NO: 6 remain unchanged and/or which comprisesone, two or three amino acid additions at the N-terminus and/orC-terminus, and which is optionally part of a fusion protein orconjugated to an agent that increases the potency and/or specificity ofthe variant, with the proviso that the variant has activitycharacteristic of the unaltered polypeptide, optionally to a greater orlesser extent.
 8. A purified or isolated variant of: (i) a polypeptideconsisting essentially of the amino acid sequence of NCEHFVTYCRYG (SEQID NO: 1), (ii) a polypeptide consisting essentially of the amino acidsequence of ENCEHFVNELRYG (SEQ ID NO: 2), (iii) a polypeptide consistingessentially of the amino acid sequence of NCEHFVNELRYG (SEQ ID NO: 3),(iv) a polypeptide consisting essentially of the amino acid sequence ofRNCEHFVAQLRYG (SEQ ID NO: 4), (v) a polypeptide consisting essentiallyof the amino acid sequence of NCEHFVAQLRYG (SEQ ID NO: 5), or (vi) apolypeptide consisting essentially of the amino acid sequence ofNCEHFVTYLRYG (SEQ ID NO: 6), which comprises one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2,amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO:4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 in SEQ ID NO: 6 remainunchanged, and/or which comprises one, two or three amino acid additionsat the N-terminus and/or C-terminus, and which is optionally part of afusion protein or conjugated to an agent that increases the potencyand/or specificity of the variant, with the proviso that the variant hasactivity characteristic of the unaltered polypeptide, optionally to agreater or lesser extent.
 9. A composition comprising (a) one or more of(i)-(vii) and (b) a carrier, excipient or adjuvant, wherein (i)-(vii)are as follows: (i) a polypeptide consisting essentially of the aminoacid sequence of NCEHFVTYCRYG (SEQ ID NO: 1), (ii) a polypeptideconsisting essentially of the amino acid sequence of ENCEHFVNELRYG (SEQID NO: 2), (iii) a polypeptide consisting essentially of the amino acidsequence of NCEHFVNELRYG (SEQ ID NO: 3), (iv) a polypeptide consistingessentially of the amino acid sequence of RNCEHFVAQLRYG (SEQ ID NO: 4),(v) a polypeptide consisting essentially of the amino acid sequence ofNCEHFVAQLRYG (SEQ ID NO: 5), (vi) a polypeptide consisting essentiallyof the amino acid sequence of NCEHFVTYLRYG (SEQ ID NO: 6), and (vii) apurified or isolated variant of one or more of (i), (ii) and (iv)-(vi),which comprises one, two or three conservative or neutral amino acidsubstitutions, provided that amino acids 1-6 and 8-12 in SEQ ID NO: 1,amino acid 1-9 and 11-13 in SEQ ID NO: 2, amino acids 1-8 and 11-13 inSEQ ID NO: 4, amino acids 1-7 and 9-12 in SEQ ID NO: 5, and amino acids1-6 and 8-12 in SEQ ID NO: 6 remain unchanged and/or which comprisesone, two or three amino acid additions at the N-terminus and/orC-terminus, and wherein any of (i)-(vii) is optionally part of a fusionprotein or conjugated to an agent that increases the potency and/orspecificity of the polypeptide or variant, and wherein the compositionoptionally further comprises an anti-cancer agent.
 10. The compositionof claim 9, which, when the composition comprises (iv), (v), (vi) and/ora purified or isolated variant of any one or more of the foregoing, thecomposition further comprises a retinoid.
 11. A composition comprising(a) one or more of (i)-(vii) and (b) a carrier, excipient or adjuvant,wherein (i)-(vii) are as follows: (i) a purified or isolated polypeptideconsisting essentially of the amino acid sequence of NCEHFVTYCRYG (SEQID NO: 1), (ii) a purified or isolated polypeptide consistingessentially of the amino acid sequence of ENCEHFVNELRYG (SEQ ID NO: 2),(iii) a purified or isolated polypeptide consisting essentially of theamino acid sequence of NCEHFVNELRYG (SEQ ID NO: 3), (iv) a purified orisolated polypeptide consisting essentially of the amino acid sequenceof RNCEHFVAQLRYG (SEQ ID NO: 4), (v) a purified or isolated polypeptideconsisting essentially of the amino acid sequence of NCEHFVAQLRYG (SEQID NO: 5), (vi) a purified or isolated polypeptide consistingessentially of the amino acid sequence of NCEHFVTYLRYG (SEQ ID NO: 6),and (vii) a purified or isolated variant of one or more of (i)-(vi),which comprises one, two or three conservative or neutral amino acidsubstitutions, provided that amino acids 8 and 9 in SEQ ID NO: 1, aminoacids 1, 8 and 9 in SEQ ID NO: 2, amino acids 7 and 8 in SEQ ID NO: 3,amino acids 1 and 8 in SEQ ID NO: 4, amino acid 7 in SEQ ID NO: 5, andamino acid 7 in SEQ ID NO: 6 remain unchanged, and/or which comprisesone, two or three amino acid additions at the N-terminus and/orC-terminus, with the proviso that the variant has activitycharacteristic of the unaltered polypeptide, optionally to a greater orlesser extent, wherein any of (i)-(vii) is optionally part of a fusionprotein or conjugated to an agent that increases the potency and/orspecificity of the polypeptide or variant thereof, and wherein thecomposition optionally further comprises an anti-cancer agent.
 12. Thecomposition of claim 11, which, when the composition comprises (iv),(v), (vi) and/or a purified or isolated variant of any one or more ofthe foregoing, the composition further comprises a retinoid.
 13. Apurified or isolated nucleic acid consisting essentially of a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 1, optionally aspart of an encoded fusion protein, and optionally in the form of avector, which is optionally targeted to a membrane receptor.
 14. Thepurified or isolated nucleic acid of claim 13, wherein the nucleic acidhas a peptidic backbone when not in the form of a vector.
 15. A purifiedor isolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 2, optionally as part ofan encoded fusion protein, and optionally in the form of a vector, whichis optionally targeted to a membrane receptor.
 16. The purified orisolated nucleic acid of claim 15, wherein the nucleic acid has apeptidic backbone when not in the form of a vector.
 17. A purified orisolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3, optionally as part ofan encoded fusion protein, and optionally in the form of a vector, whichis optionally targeted to a membrane receptor.
 18. The purified orisolated nucleic acid of claim 17, wherein the nucleic acid has apeptidic backbone when not in the form of a vector.
 19. A purified orisolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 4, optionally as part ofan encoded fusion protein, and optionally in the form of a vector, whichis optionally targeted to a membrane receptor.
 20. The purified orisolated nucleic acid of claim 19, wherein the nucleic acid has apeptidic backbone when not in the form of a vector.
 21. A purified orisolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 5, optionally as part ofan encoded fusion protein, and optionally in the form of a vector, whichis optionally targeted to a membrane receptor.
 22. The purified orisolated nucleic acid of claim 21, wherein the nucleic acid has apeptidic backbone when not in the form of a vector.
 23. A purified orisolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 6, optionally as part ofan encoded fusion protein, and optionally in the form of a vector, whichis optionally targeted to a membrane receptor.
 24. The purified orisolated nucleic acid of claim 23, wherein the nucleic acid has apeptidic backbone when not in the form of a vector.
 25. A purified orisolated variant of: (i) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of NCEHFVTYCRYG(SEQ ID NO: 1), (ii) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of ENCEHFVNELRYG(SEQ ID NO: 2), (iii) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of RNCEHFVAQLRYG(SEQ ID NO: 4), (iv) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of NCEHFVAQLRYG(SEQ ID NO: 5), or (v) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of NCEHFVTYLRYG(SEQ ID NO: 6), which comprises nucleotides encoding one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids amino acids 1-6 and 8-12 in SEQ ID NO: 1, amino acid 1-9 and 11-13in SEQ ID NO: 2, amino acids 1-8 and 11-13 in SEQ ID NO: 4, amino acids1-7 and 9-12 in SEQ ID NO: 5, and amino acids 1-6 and 8-12 in SEQ ID NO:6 remain unchanged, and/or which comprises nucleotides encoding up toone, two or three amino acid additions at the N-terminus and/orC-terminus, which is optionally part of an encoded fusion protein, andwhich is optionally in the form of a vector, which is optionallytargeted to a membrane receptor, and when the variant is not in the formof a vector, optionally has a peptidic backbone, with the proviso thatthe variant encodes a polypeptide that has activity characteristic ofthe unaltered polypeptide, optionally to a greater or lesser extent. 26.A purified or isolated variant of: (i) a nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofNCEHFVTYCRYG (SEQ ID NO: 1), (ii) a nucleic acid consisting essentiallyof a nucleotide sequence encoding the amino acid sequence ofENCEHFVNELRYG (SEQ ID NO: 2), (iii) a nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofNCEHFVNELRYG (SEQ ID NO: 3), (iv) a nucleic acid consisting essentiallyof a nucleotide sequence encoding the amino acid sequence ofRNCEHFVAQLRYG (SEQ ID NO: 4), (v) a nucleic acid consisting essentiallyof a nucleotide sequence encoding the amino acid sequence ofNCEHFVAQLRYG (SEQ ID NO: 5), or (vi) a nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofNCEHFVTYLRYG (SEQ ID NO: 6), which comprises nucleotides encoding one,two or three conservative or neutral amino acid substitutions, providedthat amino acids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQID NO: 2, amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 inSEQ ID NO: 4, amino acid 7 in SEQ ID NO: 5, and amino acids 7-9 in SEQID NO: 6 remain unchanged, and/or which comprises nucleotides encodingup to one, two or three amino acid additions at the N-terminus and/orC-terminus, which is optionally part of an encoded fusion protein, andwhich is optionally in the form of a vector, which is optionallytargeted to a membrane receptor, and when the variant is not in the formof a vector, optionally has a peptidic backbone, with the proviso thatthe variant encodes a polypeptide that has activity characteristic ofthe unaltered polypeptide, optionally to a greater or lesser extent. 27.A composition comprising (a) one or more of (i)-(vii) and (b) a carrier,excipient or adjuvant, wherein (i)-(vii) are as follows: (i) a purifiedor isolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of NCEHFVTYCRYG (SEQ ID NO: 1), (ii) apurified or isolated nucleic acid consisting essentially of a nucleotidesequence encoding the amino acid sequence of ENCEHFVNELRYG (SEQ ID NO:2), (iii) a purified or isolated nucleic acid consisting essentially ofa nucleotide sequence encoding the amino acid sequence of NCEHFVNELRYG(SEQ ID NO: 3), (iv) a purified or isolated nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofRNCEHFVAQLRYG (SEQ ID NO: 4), (v) a purified or isolated nucleic acidconsisting essentially of a nucleotide sequence encoding the amino acidsequence of NCEHFVAQLRYG (SEQ ID NO: 5), (vi) a purified or isolatednucleic acid consisting essentially of a nucleotide sequence encodingthe amino acid sequence of NCEHFVTYLRYG (SEQ ID NO: 6), and (vii) apurified or isolated variant of one or more of (i), (ii) and (iv)-(vi),which comprises nucleotides encoding one, two or three conservative orneutral amino acid substitutions, provided that amino acids 1-6 and 8-12in SEQ ID NO: 1, amino acid 1-9 and 11-13 in SEQ ID NO: 2, amino acids1-8 and 11-13 in SEQ ID NO: 4, amino acids 1-7 and 9-12 in SEQ ID NO: 5,and amino acids 1-6 and 8-12 in SEQ ID NO: 6 remain unchanged, and/orwhich comprises nucleotides encoding up to one, two or three amino acidadditions at the N-terminus and/or C-terminus, with the proviso that thevariant has activity characterisitic of the unaltered polypeptide,optionally to a greater or lesser extent, wherein any of (i)-(vii) isoptionally part of an encoded fusion protein, wherein any of (i)-(vii)is optionally in the form of a vector, which is optionally targeted to amembrane receptor, wherein, when any of (i)-(vii) is not in the form ofa vector, optionally has a peptidic backbone, and wherein thecomposition optionally further comprises an anti-cancer agent.
 28. Thecomposition of claim 27, which, when the composition comprises (iv),(v), (vi) and/or a purified or isolated variant of any one or more ofthe foregoing, the composition further comprises a retinoid.
 29. Acomposition comprising (a) one or more of (i)-(vii) and (b) a carrier,excipient or adjuvant, wherein (i)-(vii) are as follows: (i) a purifiedor isolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of NCEHFVTYCRYG (SEQ ID NO: 1), (ii) apurified or isolated nucleic acid consisting essentially of a nucleotidesequence encoding the amino acid sequence of ENCEHFVNELRYG (SEQ ID NO:2), (iii) a purified or isolated nucleic acid consisting essentially ofa nucleotide sequence encoding the amino acid sequence of NCEHFVNELRYG(SEQ ID NO: 3), (iv) a purified or isolated nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofRNCEHFVAQLRYG (SEQ ID NO: 4), (v) a purified or isolated nucleic acidconsisting essentially of a nucleotide sequence encoding the amino acidsequence of NCEHFVAQLRYG (SEQ ID NO: 5), (vi) a purified or isolatednucleic acid consisting essentially of a nucleotide sequence encodingthe amino acid sequence of NCEHFVTYLRYG (SEQ ID NO: 6), and (vii) apurified or isolated variant of one or more of (i)-(vi), which comprisesnucleotides encoding one, two or three conservative or neutral aminoacid substitutions, provided that amino acids 8 and 9 in SEQ ID NO: 1,amino acids 1, 8 and 9 in SEQ ID NO: 2, amino acids 7 and 8 in SEQ IDNO: 3, amino acids 1 and 8 in SEQ ID NO: 4, amino acid 7 in SEQ ID NO:5, and amino acid 7 in SEQ ID NO: 6 remain unchanged, and/or whichcomprises nucleotides encoding up to one, two or three amino acidadditions at the N-terminus and/or C-terminus, with the proviso that thevariant has activity characterisitic of the unaltered polypeptide,optionally to a greater or lesser extent, wherein any of (i)-(vii) isoptionally part of an encoded fusion protein, wherein any of (i)-(vii)is optionally in the form of a vector, which is optionally targeted to amembrane receptor, wherein, when any of (i)-(vii) is not in the form ofa vector, optionally has a peptidic backbone, and wherein thecomposition optionally further comprises an anti-cancer agent.
 30. Thecomposition of claim 29, which, when the composition comprises (iv),(v), (vi) and/or a purified or isolated variant of one or more of theforegoing, the composition further comprises a retinoid.
 31. A host cellcomprising: (i) a purified or isolated nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofNCEHFVTYCRYG (SEQ ID NO: 1), (ii) a purified or isolated nucleic acidconsisting essentially of a nucleotide sequence encoding the amino acidsequence of ENCEHFVNELRYG (SEQ ID NO: 2), (iii) a purified or isolatednucleic acid consisting essentially of a nucleotide sequence encodingthe amino acid sequence of NCEHFVNELRYG (SEQ ID NO: 3), (iv) a purifiedor isolated nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of RNCEHFVAQLRYG (SEQ ID NO: 4), (v) apurified or isolated nucleic acid consisting essentially of a nucleotidesequence encoding the amino acid sequence of NCEHFVAQLRYG (SEQ ID NO:5), (vi) a purified or isolated nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of NCEHFVTYLRYG(SEQ ID NO: 6), (vii) a purified or isolated variant of one or more of(i), (ii) and (iv)-(vi), which comprises nucleotides encoding one, twoor three conservative or neutral amino acid substitutions, provided thatamino acids amino acids 1-6 and 8-12 in SEQ ID NO: 1, amino acid 1-9 and11-13 in SEQ ID NO: 2, amino acids 1-8 and 11-13 in SEQ ID NO: 4, aminoacids 1-7 and 9-12 in SEQ ID NO: 5, and amino acids 1-6 and 8-12 in SEQID NO: 6 remain unchanged, and/or which comprises nucleotides encodingup to one, two or three amino acid additions at the N-terminus and/orC-terminus, and (viii) a purified or isolated variant of one or more of(i)-(vi), which comprises nucleotides encoding one, two or threeconservative or neutral amino acid substitutions, provided thatnucleotides encoding amino acids 8 and 9 in SEQ ID NO: 1, amino acids 1,8 and 9 in SEQ ID NO: 2, amino acids 7 and 8 in SEQ ID NO: 3, aminoacids 1 and 8 in SEQ ID NO: 4, amino acid 7 in SEQ ID NO: 5, and aminoacid 7 in SEQ ID NO: 6 remain unchanged, and/or which comprisesnucleotides encoding up to one, two or three amino acid additions at theN-terminus and/or C-terminus, with the proviso that the variant hasactivity characterisitic of the unaltered polypeptide, optionally to agreater or lesser extent, wherein any of (i)-(viii) is optionally partof an encoded fusion protein, wherein any of (i)-(viii) is optionally inthe form of a vector, which is optionally targeted to a membranereceptor, and wherein, when any of (i)-(viii) is not in the form of avector, optionally has a peptidic backbone.
 32. A method of inhibitingcancerous cell growth, which method comprises contacting a collection ofcancerous cells with a cell growth-inhibiting effective amount of one ormore of the following: (i) at least one purified or isolatedpolypeptide, which can be the same or different, selected from the groupconsisting of: (a) a polypeptide consisting essentially of the aminoacid sequence of SEQ ID NO: 2, (b) a polypeptide consisting essentiallyof the amino acid sequence of SEQ ID NO: 3, (c) a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO: 4, (d) apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO: 5, (e) a polypeptide consisting essentially of the amino acidsequence of SEQ ID NO: 6, (f) a purified or isolated variant of one ormore of (a) and (c)-(e), which comprises nucleotides encoding one, twoor three conservative or neutral amino acid substitutions, provided thatamino acids amino acids 1-6 and 8-12 in SEQ ID NO: 1, amino acid 1-9 and11-13 in SEQ ID NO: 2, amino acids 1-8 and 11-13 in SEQ ID NO: 4, aminoacids 1-7 and 9-12 in SEQ ID NO: 5, and amino acids 1-6 and 8-12 in SEQID NO: 6 remain unchanged, and/or which comprises nucleotides encodingup to one, two or three amino acid additions at the N-terminus and/orC-terminus, with the proviso that the variant has activitycharacteristic of the unaltered polypeptide, optionally to a greater orlesser extent, and (g) a purified or isolated variant of any of theaforementioned polypeptides (a)-(e), which comprises one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2,amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO:4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 in SEQ ID NO: 6 remainunchanged, and/or which comprises one, two or three amino acid additionsat the N-terminus and/or C-terminus, with the proviso that the varianthas activity characteristic of the unaltered polypeptide, optionally toa greater or lesser extent, wherein any of (a)-(g) is optionally part ofa fusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide or variant thereof, (ii) acomposition comprising (i) and a carrier, excipient or adjuvant, and,optionally, an anti-cancer agent, and, when the polypeptide consistsessentially of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 orSEQ ID NO: 6, optionally, a retinoid, (iii) at least one purified orisolated nucleic acid, which can be the same or different, selected fromthe group consisting of: (a′) a nucleic acid consisting essentially of anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2,(b′) a nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3, (c′) a nucleic acidconsisting essentially of a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 4, (d′) a nucleic acid consisting essentially ofa nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5,(e′) a nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 6, (f′) a nucleic acidconsisting essentially of a nucleotide sequence encoding a variant ofone or more of (a′) and (c′)-(e′), which comprises nucleotides encodingone, two or three conservative or neutral amino acid substitutions,provided that amino acids amino acids 1-6 and 8-12 in SEQ ID NO: 1,amino acid 1-9 and 11-13 in SEQ ID NO: 2, amino acids 1-8 and 11-13 inSEQ ID NO: 4, amino acids 1-7 and 9-12 in SEQ ID NO: 5, and amino acids1-6 and 8-12 in SEQ ID NO: 6 remain unchanged, and/or which comprisesnucleotides encoding up to one, two or three amino acid additions at theN-terminus and/or C-terminus, and (g′) a nucleic acid consistingessentially of a nucleotide sequence encoding a variant of one or moreof (a′)-(f′), which comprises nucleotides encoding one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2,amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO:4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 in SEQ ID NO: 6 remainunchanged, and/or which comprises nucleotides encoding up to one, two orthree amino acid additions at the N-terminus and/or C-terminus, with theproviso that the variant has activity characteristic of the unalteredpolypeptide, optionally to a greater or lesser extent, wherein any of(a′)-(g′) is optionally part of an encoded fusion protein, wherein anyof (a′)-(g′) is optionally in the form of a vector, which is optionallytargeted to a membrane receptor, wherein, when any of (a′)-(g′) is notin the form of a vector, optionally has a peptidic backbone, and (iv) acomposition comprising (iii) and a carrier, excipient or adjuvant, and,optionally, an anti-cancer agent, and, when the nucleic acid encodes theamino acid of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, optionallyretinoic acid, wherein the method optionally further comprisesseparately contacting the collection of cells with an anti-cancer agent,which can be the same or different, in the same manner or a differentmanner, surgical removal of the collection of cancerous cells when invivo, and/or radiation, whereupon the growth of the collection ofcancerous cells is inhibited.
 33. The method of claim 32, wherein thecollection of cells is in vivo.
 34. The method of claim 33, wherein thecollection of cells is a melanoma.
 35. The method of claim 33, whereinthe collection of cells is in a mammal.
 36. A method of modulating geneexpression, which method comprises contacting a collection of cells witha gene expression-modulating effective amount of one or more of thefollowing: (i) at least one purified or isolated nucleic acid, which canbe the same or different, selected from the group consisting of: (a) anucleic acid consisting essentially of a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO: 1, (b) a nucleic acid consistingessentially of a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 2, (c) a nucleic acid consisting essentially of a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 3, (d) a nucleicacid consisting essentially of a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 4, (e) a nucleic acid consisting essentiallyof a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:5, (f) a nucleic acid consisting essentially of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 6, (g) a nucleic acidconsisting essentially of a nucleotide sequence encoding a variant ofone or more of (a), (b) and (d)-(f), which comprises nucleotidesencoding one, two or three conservative or neutral amino acidsubstitutions, provided that amino acids amino acids 1-6 and 8-12 in SEQID NO: 1, amino acid 1-9 and 11-13 in SEQ ID NO: 2, amino acids 1-8 and11-13 in SEQ ID NO: 4, amino acids 1-7 and 9-12 in SEQ ID NO: 5, andamino acids 1-6 and 8-12 in SEQ ID NO: 6 remain unchanged, and/or whichcomprises nucleotides encoding up to one, two or three amino acidadditions at the N-terminus and/or C-terminus, with the proviso that thevariant has activity characteristic of the unaltered polypeptide,optionally to a greater or lesser extent, and (h) a nucleic acidconsisting essentially of a nucleotide sequence encoding a variant ofone or more of (a)-(f), which comprises nucleotides encoding one, two orthree conservative or neutral amino acid substitutions, provided thatamino acids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ IDNO: 2, amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQID NO: 4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 in SEQ ID NO: 6remain unchanged, and/or which comprises nucleotides encoding up to one,two or three amino acid additions at the N-terminus and/or C-terminus,with the proviso that the variant has activity characterisitic of theunaltered polypeptide, optionally to a greater or lesser extent, whereinsaid purified or isolated nucleic acid has a peptidic backbone, and (ii)a composition comprising (i) and a carrier, excipient or adjuvant,whereupon gene expression in the collection of cells is modulated. 37.The method of claim 36, wherein, when the nucleic acid is (d), (e), (f)or a variant thereof in accordance with (f) or (g), the compositionfurther comprises a retinoid.
 38. The method of claim 36, wherein thecollection of cells is in vivo.
 39. The method of claim 38, wherein thecollection of cells is cancerous.
 40. The method of claim 39, whereinthe collection of cells is a melanoma.
 41. The method of claim 40,wherein the gene is retinoic acid receptor β or high mobility group I(Y) and the expression of the gene is upregulated.
 42. The method ofclaim 38, wherein the collection of cells is in a mammal.
 43. A methodof enhancing the immune response-inducing effect of a vaccine, whichmethod comprises adding to the vaccine at least one purified or isolatedpolypeptide, which can be the same or different, selected from the groupconsisting of: (a) a polypeptide consisting essentially of the aminoacid sequence of SEQ ID NO: 1, (b) a polypeptide consisting essentiallyof the amino acid sequence of SEQ ID NO: 2, (c) a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO: 3, (d) apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO: 4, (e) a polypeptide consisting essentially of the amino acidsequence of SEQ ID NO: 5, (f) a polypeptide consisting essentially ofthe amino acid sequence of SEQ ID NO: 6, (g) a variant of any of thepolypeptides of (a), (b) and (d)-(f), which comprises one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids amino acids 1-6 and 8-12 in SEQ ID NO: 1, amino acid 1-9 and 11-13in SEQ ID NO: 2, amino acids 1-8 and 11-13 in SEQ ID NO: 4, amino acids1-7 and 9-12 in SEQ ID NO: 5, and amino acids 1-6 and 8-12 in SEQ ID NO:6 remain unchanged, and/or which comprises one, two or three amino acidadditions at the N-terminus and/or C-terminus, with the proviso that thevariant has activity characteristic of the unaltered polypeptide,optionally to a greater or lesser extent, and (h) a variant of any ofthe aforementioned polypeptides, which comprises one, two or threeconservative or neutral amino acid substitutions, provided that aminoacids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2,amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO:4, amino acid 7 in SEQ ID NO: 5, and amino acid 7 in SEQ ID NO: 6 remainunchanged, and/or which comprises one, two or three amino acid additionsat the N-terminus and/or C-terminus, with the proviso that the varianthas activity characteristic of the unaltered polypeptide, optionally toa greater or lesser extent, wherein any of (a)-(g) is optionally part ofa fusion protein or conjugated to an agent that increases the potencyand/or specificity of the polypeptide or variant thereof, whereupon theimmune response-inducing effect of the vaccine is enhanced.